Antitumor agent

ABSTRACT

An antitumor agent of the present invention comprises, as an effective ingredient, lipoteichoic acid which is extracted from gram-positive bacterial cells.

FIELD OF THE INVENTION

This invention relates to an antitumor agent comprising lipoteichoicacids (hereafter referred to AS LTA) as an effective ingredient.

PRIOR ART

LTA is a group of amphipathic substances found in gram-positivebacterial cells. LTA consists of a polymer chain of polyglycerophosphate(PGP) as a backbone structure and glycolipids of cytoplasmic membraneorigin (A. J. Wicken et al., Biological Properties of LipoteichoicAcids, Microbiology, pp 360-365, 1977).

Electronmicroscopic analysis by ferritin labelled antibody techniquereveals that the one end of the LTA is linked to cytoplasmic membraneglycolipid while the other end of the LTA extends to the cell outersurface of the bacteria through the cell wall peptidoglycan layer.

Examples of gram positive bacteria having LTA are those belonging tosuch genera as Streptococcus, Micrococcus, Lactobacillus,Staphylococcus, Bacillus and Listeria. The structures of LTA maypartially differ among bacteria of different genera or even amongdifferent species of the same genus. For example, differences in length(25-30) of PGP chain, or in number or type of sugar residues in theglycolipid moiety are known.

LTA has not been fully studied with respect to its immunopharmacologicalactivities. In particular, the use of LTA for treatment of tumorpatients has never been reported.

SUMMARY OF THE INVENTION

The present inventors have found that LTA has an excellent antitumoractivity without causing any side effects in animals.

The main object of the present invention is to provide an antitumoragent comprising LTA as an effective ingredient together with a suitablediluent or carrier.

Other objects of the invention will become clear by reading thefollowing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the antitumor activity of LTA in Example 1.

FIG. 2 shows the antitumor activity of LTA in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

Partial structural differences of LTA do not have serious influence onits biological activities. Thus LTA from any species or genera ofgram-positive bacteria has good possibility to serve as an antitumoragent of the present invention.

For example, LTA obtained from Streptococcus pyogenes, which contains0.5 mole alaine, 0.82 mole phosphoric acid, 0.05 mole glucose and 0.012mole fatty acids relative to 1 mole of glycerol, may be convenientlyemployed.

Examples of bacterial strains having such LTA and which may be used inthe present invention are as follows:

Streptococcus faecalis

Streptococcus pyogenes

Streptococcus mutans

Streptococcus lactis

Streptococcus equisimilis (FERM-P 4059)

Streptococcus sanguis

Staphylococcus aureus

Staphylococcus epidermidis

Lactobacillus plantarum

Lactobacillus fermentum

Lactobacillus casei

Listeria monocytogenes.

The preparation of LTA from whole cells, or a cell envelope fraction ofthese bacteria may be obtained, for example, according to the methoddescribed by Beachey et al. (Infect. Immun., 23, 618-625, 1979).

LTA is soluble in both water and a lipophilic medium because of itsamphipathic property. Therefore, it can be formulated by a conventionalformulation process into any desired form such as an oral agent or aparentheral agent containing a desired amount of the active ingredient.

Examples of carriers which may be used for formulating such agents arecrystalline cellulose, corn starch, sugars such as glucose and lactose,magnesium stearate and calcium stearate.

In general, for good antitumor effects to be obtained in man, 0.1-100mg, preferably 1-20 mg of LTA is administered to an adult as a dailydosage unit in the agent of the present invention.

PREPARATION EXAMPLE

Streptococcus pyogenes SV strain (ATCC 21059) was cultured in ToddHewitt broth (Difco, USA) overnight. Cells were collected and disruptedby a Braun cell homogenizer. A fraction of cell envelopes was collectedby centrifugation and it was suspended in distilled water at 10 mg (dryweight)/ml. An equal volume of 95% phenol was added to the suspension,and extraction of LTA was conducted by gentle stirring at a temperaturebetween 4° C. and room temperature for an hour. The water phase wasseparated by centrifugation (18000×g) for 30 minutes. An equal volume ofwater was added to the remaining phenol phase to repeat the extractiontwice in the same manner. All of the water phases were combined andafter being fully dialysed against distilled water, the combined waterphase was lyophilized to obtain a crude LTA preparation.

The crude LTA was dissolved in 0.2M ammonium acetate at a concentrationof 50 mg (dry weight)/ml, which was then applied to a gel filtrationcolumn (2.6 φ×87 cm) of Sepharose 6B (Pharmacia Co., Sweden) and wasfractionated with 0.2M ammonium acetate as an eluant, whereby the crudeLTA was purified. Each effluent was monitored by its activity tosensitize sheep red blood cells (SRBC) to be agglutinated by anti-PGSserum, and by a colorimetric determination of phosphorus to determinethe localization of LTA.

EXAMPLE 1 Anti-tumor effect of streptococcal LTA on solid-type Meth-Afibrosarcoma

Groups of 5 female, 6 week-old BALB/c mice (purchased from Charles RiverJapan Inc., Japan) were transplated at the abdominal regionintradermally with a suspension of Meth-A fibrosarcoma cells (2×10⁵/mouse). LTA obtained in the Preparation Example was dissolved inphysiological saline and 0.2 ml of the solution was intraperitoneallyadministered daily for four successive days from the day after the tumorinoculation. Mice of the control group were administered withphysiological saline (0.2 ml) in place of the LTA solution.

To evaluate antitumor activity, tumor size was determined in terms of√length×width at intervals and growth inhibition rate was obtained as aratio of average tumor sizes in the LTA group and the control group.

The result is shown in FIG. 1. A significant antitumor effect wasobserved 22 days after the transplantation in the group administeredwith 10 μg and 2 μg of LTA.

EXAMPLE 2 Anti-tumor effect of streptococcal LTA on ascites type Meth-Afibrosarcoma

Groups of 5 mice of the same strain as used in Example 1 weretransplanted in their peritoneal cavities with 2×10⁵ cells of Meth-Afibrosarcoma.

LTA solution obtained in the preparation Example was administered intomice in the same method as described in Example 1.

The anti-tumor effect was expressed by the following formula: ##EQU1##As shown in FIG. 2, the anti-tumor effect of LTA was definitely observedin mice with ascites type of Meth-A fibrosarcoma. In addition, two outof five mice in each of the test groups which are given the injection of10 μg or 50 μg of LTA were still surviving 60 days after the tumorinoculation, while all mice in the control group died within 24 days.

EXAMPLE 3 Anti-tumor effect of Lactobacillary LTA on solid-type Meth-Afibrosarcoma

Anti-tumor effect was studied as described in Example 1 using LTA whichwas obtained from Lactobacillus plantarum according to the methoddescribed in the Preparation Example. As shown in FIG. 3, LTA from L.plantarum also has an antitumor effect on solid-type Meth-Afibrosarcoma.

The chemical composition of the LTA is shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Chemical Composition of LTA-fraction                                          of L. plantarum ATCC 8014                                                                  LTA-fraction                                                     Component      %           Mole ratio                                         ______________________________________                                        Hexose         15.2        0.27                                               Glycerol       28.4        1.00                                               Phosphorus     34.4        1.15                                               Protein        --                                                             Pentose        0.16         0.003                                             Fatty acids    7.3         0.08                                               Methylpentose  2.9         0.05                                               Alanine        Not determined                                                 ______________________________________                                    

The foregoing Examples 1-3 clearly show the definite anti-tumor effectof LTA.

As one of the approaches to studying the mechanism of the anti-tumoreffect of LTA, the present inventors have examined a possible activityof LTA to induce tumor necrotizing factor (TNF), and have found that LTAis a potent inducer of TNF as described in Example 4. TNF is a substancefound in the serum of LPS treated animals which had been primed byprevious injection of B.C.G. or Propionibacterium acnes TNF causesnecrosis of some animal tumors in vivo, and is cytotoxic or cytostaticto tumor cell lines of animal and human origin in vitro. The abovefinding suggests that the anti-tumor effect of LTA may be at leastpartly due to the TNF-inducing activity of LTA.

EXAMPLE 4 Induction of TNF by LTA in mice primed with P. acnes (1)Preparation of TNF

A group of 8 female, 5 week-old ICR mice (purchased from Charles RiverJapan Inc.) were given an intraperitoneal injection of formalin-killedcells (1.5 mg/mouse) of P. acnes.

On the 11th day, the mice received an intravenous injection of LTA (100μg/mouse) obtained in the Preparation Example. Two hours later, the micewere bled and a serum was prepared by a conventional method. Serumspecimens were prepared also from groups treated only with P. acnes orLTA as well as from a non-treated group. Each serum was centrifuged(39,000×g) for an hour. The upper one third portion of the supernatantwas discarded and TNF activity of the serum was measured using the lowertwo thirds portion of the supernatant (hereafter referred to asLTA-induced TNF).

(2) Cytotoxic activity of LTA-induced TNF on L-cells

A suspension of L-929 cell (1×10⁵ cells/ml) in RPMI-1640 mediumsupplemented with 10% FCS (fetal calf serum) was distributed in amicroplate having 96 wells (80 μl/well), and the cells were incubatedfor 3 hours at 37° C. under at atmosphere of 5% CO₂ -air. Then 100 μl ofthe TNF sample obtained in above (1) which was serially 5 fold dilutedwith an RPMI-1640 medium containing FCS and 20 μl ³ H-thymidine solution(1 μC_(i)) was added to the cells in each well. The cells were thencultured at 37° C. under 5% CO₂ -air.

The medium was discarded after 48 hours and the L-929 cells werecollected with a cell harvester after being released from the plate bytrypsin-EDTA method so as to enable the radioactivity (c.p.m) of ³H-thymidine uptaken in the cells to be counted.

The cytotoxic effect of streptococcal LTA-induced TNF on L-929 cells interms of the activity to inhibit thymidine uptake is shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Cytotoxic effect of streptococcal                                             LTA-induced TNF on L-cells                                                    Serum specimen                                                                from mice                                                                     treated with.sup.(a)                                                          P. acnes                                                                             LTA     .sup.3 H--Thymidine uptake.sup.(b)                                                             % Inhibition.sup.(c)                          ______________________________________                                        -      -       22.632 ± 363  -3.0                                          +      -       20.740 ± 668  5.5                                           -      +       20.559 ± 357  6.4                                           +      +       5.766 ± 88    73.7                                          RPMI-1640  21.955 ± 540  0                                                 with 10% FCS                                                                  ______________________________________                                         .sup.(a) Serum samples diluted 1:200 with RPMI1640 containing 10% FCS wer     submitted to the assay.                                                       .sup.(b)3 H--thymidine uptake was expressed as mean ± standard error       (S.E.) in triplicate. Background count (235 c.p.m.) was subtrated from        each determination.                                                           .sup.(c) Percent inhibition of thymidine uptake was calculated by the         equation:                                                                     ##STR1##                                                                 

Several other mouse tumor cell lines were examined for theirsusceptibilit to the cytotoxic effect of LTA-induced TNF in the samesystem as described above. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Cytotoxic effect of                                                           Streptococcal LTA-induced TNF                                                 on various tumor cell lines                                                                Growth inhibition (%)                                                         P. acnes-LTA                                                     Cell line      1/100.sup.(a)                                                                          1/1000.sup.(a)                                        ______________________________________                                        Meth-A         66.5**   43.7**                                                EL-4           51.4**   9.8                                                   BAMC-1         17.3     12.7                                                  L1210          39.3**   -2.7                                                  P388           37.6**   6.7                                                   MH-134         30.9     6.5                                                   C1498          54.2*    8.8                                                   3LL            73.2***  54.8***                                               B16            20.7     5.6                                                   ______________________________________                                         .sup.(a) Serum dilution ratio.                                                ***P < 0.001, **P < 0.01, *P < 0.05                                      

(3) Cytocidal effect of Streptococcal LTA-induced TNF on L-cells

A suspension of L-929 cells (1×10⁵ cells/ml) in RPMI-1640 mediumsupplemented with 10% FCS was distributed in a microplate having 24wells (0.5 ml/well), and the cells were incubated for 3 hours at 37° C.under 5% CO₂ -air. Then, to the cells in each well was added 0.5 ml ofthe TNF sample obtained in above (1) diluted 1:100 with RPMI-1640containing 10% FCS. The culture was then continued for a further 48hours.

At the end of the cultivation, the numbers of viable and dead cells inthe medium and in the cell suspension released from the microplate bytrypsin-EDTA method were counted under a phase contrast microscope.

                  TABLE 4                                                         ______________________________________                                        Cytocidal effect of                                                           streptococcal LTA-induced                                                     TNF on L-cells                                                                Serum specimen                                                                from mice   Number of viable cells/                                           treated with.sup.(a)                                                                      Number of dead cells.sup.(b)                                                                   Cytotoxicity.sup.(c)                             P. acnes                                                                              LTA     (× 10.sup.-4 /ml)                                                                        (%)                                          ______________________________________                                        -       -       214/7            3.2                                          +       -       208/11           5.0                                          -       +       206/12           5.5                                          +       +        3/87            96.7                                         RPMI-1640                                                                             215/8   3.6                                                           with 10% FCS                                                                  ______________________________________                                         .sup.(a) Samples were 100 fold diluted with RPMI1640 containing 10% FCS,      and submitted to the assay.                                                   .sup.(b) Values are the average of duplicate counts.                          .sup.(c) Cytotoxicity was calculated by the equation:                         ##STR2##                                                                 

(4) Induction of tumor necrosis by Streptococcal LTA-induced TNF

Necrosis of tumor mass caused by LTA-induced TNF was studied accordingto the method of Carswell et al. (Proc. Nat. Acad. Sci. USA, 72 (9),3666-3670, 1975). A group of 4 female, 5 week-old BALB/c mice (CharlesRiver Japan Inc.) were given an intradermal implantation of 2×10⁵cells/mouse of Meth-A fibrosarcoma. Seven days after thetransplantation, when the tumor mass reached 7-8 mm in diameter, 0.3 mlof the TNF sample obtained in above (1) was injected twice from the tailvein at a time interval for 3 hours.

Twenty-four hours after the second administration, necrosis of the tumorwas evaluated by the grading system of Carswell et al. (ibid.). As shownin Table 5, the injection of serum of mice treated with P. acnes and LTAcaused the necrosis of established-sarcoma in all mice.

                  TABLE 5                                                         ______________________________________                                        Necrosis of sarcoma Meth-A                                                    by streptococcal LTA-induced TNF                                              Serum specimen from                                                                          Necrotic response.sup.(a)                                      mice treated with                                                                            (number of mice)                                               P. acnes LTA       -     +      ++   +++                                      ______________________________________                                        -        -         4     0      0    0                                        +        -         4     0      0    0                                        -        +         4     0      0    0                                        +        +         0     2      2    0                                        RPMI-1640 with 10% FCS                                                                       4     0        0    0                                          ______________________________________                                         .sup.(a) The grading system of Carswell et al. was adopted as follows:        - . . . no observable change                                                  + . . . 25-50% portion of tumor mass underwent necrosis                       ++ . . . 50-75% portion of tumor mass underwent necrosis                      +++ . . . more than 75% portion of tumor mass underwent necrosis         

EXAMPLE 5 Lethal toxicity of streptococcal LTA in mice

LTA obtained in the Preparation Example was given by intraperitonealinjection at varying doses of 125-40,000 μg/mouse to groups of 4 female,5 week-old BALB/c mice (Charles River Japan Inc.) with or withoutprevious priming with P. acnes. The results are shown in Table 6. Thedata also include lethal toxicity of an endotoxic lipopolysaccharide(LPS, prepared from Salmonella enteritidis, Difco Lipopolysaccharide W,USA) as a reference.

                  TABLE 6                                                         ______________________________________                                        Lethal toxicity of streptococcal LTA by                                       intraperitoneal injection                                                            Normal mice P. acnes primed mice.sup.(a)                               Dose     LTA      LPS      LTA      LPS                                       (μg/mouse)                                                                          (dead.sup.(b) /total)                                                                       (dead.sup.(b) /total)                                  ______________________________________                                        40000    0/4                                                                  20000    0/4                                                                  2000     0/4                                                                  1000     NT.sup.(c)                                                                             4/4      0/4                                                500      NT.sup.(c)                                                                             4/4      0/4                                                250      NT.sup.(c)                                                                             2/4      0/4                                                125      0/4      0/4                                                         12.5                                4/4                                       6.25                                4/4                                       3.13                                4/4                                       1.56                                3/4                                       0.8                                 2/4                                       0.4                                 0/4                                       ______________________________________                                         .sup.(a) Priming by intraperitoneal administration of formalinkilled P.       acnes (1.5 mg/mouse) was conducted 12 days before LTA injection.              .sup.(b) The number of dead animals was scored 3 days after LTA injection     .sup.(c) NT: Not tested                                                  

None of the normal mice died by the injection of 40,000 μg of LTA, while500 μg and 250 μg of a reference LPS killed all and half of each groupof 4 test mice, respectively.

With P. acnes-primed mice under the experimental condition adopted forTNF production, all of 4 test mice receiving 3.13 μg of LPS were killed.In sharp contrast with this, no death was scored with those injectedwith as high a dose as 1,000 μg of LTA. No signs of toxocities(diarrhea, ataxic gait) were observed in any of the mice administeredwith LTA.

Formulation Example

LTA injections

LTA (0.5 g) was dissolved in distilled water together with 10 g ofmannitol and the volume was adjusted to 200 ml. After a filtration ofthe solution through a filter membrane (millipore filter®, 0.22 μm), 2ml aliquots of the filtrate were placed in vials under sterilizedconditions, followed by lyophilization by a conventional method. Theresulting vials, each containing 5 mg of LTA, can be used for injectionafter dissolving in 2 ml of distilled water or physiological saline forinjection.

LTA capsules

LTA (15 g) and mannitol (55 g) were mixed homogeneously. 70 mg portionsof the mixed powder were filled in No. 4 gelatin capsules of theJapanese Pharmacopoeia. The resulting capsules, containing 15 mg of LTAin each capsule, passed Disintegration Test for Capsules in accordancewith the Japanese Pharmacopoeia.

We claim:
 1. A method for inducing the production of TNF in a mammalcomprising administering to the mammal a sufficient amount oflipoteichoic acid (LTA) to induce TNF in the mammal.
 2. The method ofclaim 1 wherein the LTA is administered in dosage units containing fromabout 0.1 to about 100 mg LTA per dosage unit.
 3. The method of claim 2wherein the LTA is administered in dosage units containing from about 1to about 20 mg of LTA per dosage unit.
 4. The method of claim 1 whereinthe LTA is obtained from Streptococcus pyogenes.
 5. The method of claim1 wherein the LTA is obtained from Lactobacillus plantarum.
 6. Themethod of claim 1 wherein the LTA is administered orally.
 7. The methodof claims 1 wherein the LTA is administered intraperitoneally.
 8. Themethod of claim 1 wherein the LTA is administered in combination with apharmaceutically acceptable diluent or carrier selected from the groupconsisting of lactose, crystalline cellulose, corn starch, glucose,magnesium stearate, and calcium stearate.
 9. The method of claim 1wherein the LTA is obtained from bacteria selected from the groupconsisting of Streptococcus faecalis, Streptococcus mutans,Streptococcus lactis, Streptococcus equisimilis, Streptococcus sanguis,Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillusfermentum, Lactobacillus casei, and Listera monocytogenes.
 10. A methodof treating solid or ascites Meth-A-fibrosarcoma in a mammal comprisingadministering to the mammal an effective amount of LTA.
 11. A method oftreating solid or ascites tumors in a mammal comprising administering tothe mammal an effective amount of LTA.